Molecular reaction mechanisms of heterotrimeric G-Proteins

异源三聚体G蛋白是细胞中信号传导通路的中心开关。这些通常是通过G蛋白偶联受体（GPCR），催化从GDP到GTP的交换。这导致异源三聚体复合物分解成其亚基并激活重要的信号传导途径。它们再次被Galpha-subunit内的GTP水解关闭。GPCR目前由世界各地的许多研究小组进行研究，特别是由于其在药理学中的重要性。相比之下，我们将研究较少详细研究的水解的α-蛋白质的广泛的，综合的方法，使用时间分辨傅立叶变换红外光谱差光谱和生物分子模拟。α亚基由G结构域和一个额外的全α结构域组成，G结构域在小GTP酶中也是保守的。从X射线晶体学中获得的几种Galalpha蛋白质结构已经可用。然而，使用了不可水解的GTP类似物，这严重干扰了催化中心。互补的，时间分辨FTIR光谱可以解决蛋白质在生理条件下的动力学及其与原子细节的相互作用，使用天然核苷酸。我们已经成功地将这种方法应用于许多小型GTP。在我们的初步工作中，我们也能够将这种方法转移到Alpha蛋白质中。为了从红外光谱中解码详细的分子信息，还应用了QM/MM计算。在这里，我们想阐明催化重要氨基酸的作用。除了谷氨酰胺，稳定亲核水，特别是催化精氨酸的作用将被确定。与小GTP酶相反，后者是催化中心内的固有残基。此外，我们想了解与小GTP酶的GAP不同，RGS蛋白如何能够进一步加速水解，而不与核苷酸直接相互作用。这调节信号通路的中断。除了野生型蛋白质，我们还想特别确定由精氨酸和谷氨酰胺突变诱导的蛋白质功能障碍。这些突变在严重疾病如McCune-Albright综合征和癌症中起重要作用。我们将从抑制性Gi和激活性Gs开始研究。稍后计划研究Gq和Gt，以阐明机制的改变和相似性。进一步的疾病如霍乱和百日咳是由毒素介导的，通过ADP-核糖基化修饰α-蛋白。将详细研究这种修饰的机制。

Heterotrimeric G-proteins are central switches within signalling pathways in cells. These are usually switched on via G-protein coupled receptors (GPCR), which catalyse the exchange from GDP to GTP. This causes the breakup of the heterotrimeric complex into its subunits and activates important signalling pathways. They are switched off again by GTP hydrolysis within the Galpha-subunit. GPCRs are currently investigated by numerous research groups worldwide, especially due to their importance in pharmacology. In contrast, we will investigate the less detailed studied hydrolysis of Galpha-proteins by a broad, integrative approach using time-resolved FTIR difference-spectroscopy and biomolecular simulations. The Galpha-subunits consist of the G-domain, conserved also in small GTPases, and an additional all-alpha domain. Several protein structures of Galpha obtained from X-ray crystallography are already available. However, non-hydrolysable GTP analogues were used, which interfere seriously with the catalytic centre. Complementary, time-resolved FTIR-spectroscopy can resolve the dynamics of the protein under physiological conditions and its interaction with atomic detail using the natural nucleotide. We have applied this approach already successfully for many small GTPases. In our preliminary work, we were able to transfer this approach also to Galpha-proteins. In order to decode detailed molecular information from the IR spectra, QM/MM calculations are applied in addition. Here we want to elucidate the role of the catalytically important amino acids. Besides a glutamine, stabilizing the nucleophilic water, in particular the role of the catalytic arginine will be determined. The latter is, in contrast to small GTPases, an intrinsic residue within the catalytic centre. Further, we want to understand how RGS-proteins, unlike GAPs of small GTPases, are able to further accelerate hydrolysis, without direct interaction with the nucleotide. This regulates the interruption of signalling pathways. Besides wildtype protein, we want to determine especially dysfunctions of the protein induced by mutations of the arginine and the glutamine. These mutations play an important role in severe diseases as the McCune-Albright syndrome and cancer. We will start our investigations with the inhibitory Gi and the activating Gs. Later it is planned to investigate Gq and Gt, to elucidate mechanistic alterations and similarities. Further diseases like cholera and pertussis are mediated by toxins, modifying Galpha-proteins by ADP-ribosylation. The mechanism of this modification will be investigated in detail.